2015-03-31T22:07:46ZPK/PD modelling of combed-shaped PEGylated salmon calcitonin conjugates of differing molecular weightshttp://hdl.handle.net/10197/3020
PK/PD modelling of combed-shaped PEGylated salmon calcitonin conjugates of differing molecular weights
Ryan, Sinead M.; Frías, Jesús M.; Wang, Xuexuan; Sayers, Claire T.; Haddleton, David M.; Brayden, David James
Salmon calcitonin (sCT) was conjugated via cysteine-1 to novel comb-shaped end-functionalised (poly(PEG) methyl ether methacrylate) (sCT-P) polymers, to yield conjugates of total molecular weights (MW) inclusive of sCT: 6.5, 9.5, 23 and 40 kDa. The conjugates were characterised by HPLC and their in vitro and in vivo bioactivity was measured by cAMP assay on human T47D cells and following intravenous (i.v.) injection to rats, respectively. Stability against endopeptidases, rat serum and liver homogenates was assessed. There were linear and exponential relationships between conjugate MW with potency and efficacy respectively, however the largest MW conjugate still retained 70% of Emax and an EC50 of 3.7 nM. In vivo, while free sCT and the conjugates reduced serum [calcium] to a maximum of 15–30% over 240 min, the half-life (T1/2) was increased and the area under the curve (AUC) was extended in proportion to conjugate MW. Likewise, the polymer conferred protection on sCT against attack by trypsin, chymotrypsin, elastase, rat serum and liver homogenates, with the best protection afforded by sCT-P (40 kDa). Mathematical modelling accurately predicted the MW relationships to in vitro efficacy, potency, in vivo PK and enzymatic stability. With a significant increase in T1/2 for sCT, the 40 kDa MW comb-shaped PEG conjugate of sCT may have potential as a long-acting injectable formulation.
2011-01-20T00:00:00ZHigh content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogshttp://hdl.handle.net/10197/3018
High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs
Walsh, Edwin G.; Maher, Sam; Devocelle, Marc; O'Brien, Peter J.; Baird, Alan W.; Brayden, David James
Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates
and/or food additives. They are present in many organisms including bacteria, insects, fish and
mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current
limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial
action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is
reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs
is required in order to predict potential failure at a later stage of development.Wedescribe a high-content
analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical
drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the
actions of fluorescence microscopy with automated cell analysis software in order to understand multiple
changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic-helical peptide, melittin, and selected structural analogs. The data shows that structural modification
of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.
2011-08-01T00:00:00ZHigh content analysis of cytotoxic effects of pDMAEMA on human intestinal epithelial and monocyte cultureshttp://hdl.handle.net/10197/3009
High content analysis of cytotoxic effects of pDMAEMA on human intestinal epithelial and monocyte cultures
Rawlinson, Lee-Anne Betty; O'Brien, Peter J.; Brayden, David James
Poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) is a cationic polymer with potential as an antimicrobial agent and as a non-viral gene delivery vector. The aim was to further elucidate the cytotoxicity of a selected pDMAEMA low molecular weight (MW) polymer against human U937 monocytes and Caco-2 intestinal epithelial cells using a novel multi-parameter high content analysis (HCA) assay and to investigate histological effects on isolated rat intestinal mucosae. Seven parameters of cytotoxicity were measured: nuclear intensity (NI), nuclear area (NA), intracellular calcium ([Ca2+]i), mitochondrial membrane potential (MMP), plasma membrane permeability (PMP), cell number (CN) and phospholipidosis. Histological effects of pDMAEMA on excised rat ileal and colonic mucosae were investigated in Ussing chambers. Following 24-72 hours exposure, 25-50 µg/ml pDMAEMA induced necrosis in U937 cells, while 100-250 µg/ml induced apoptosis in Caco-2. pDMAEMA increased NA and NI and decreased [Ca2+]i, PMP, MMP and CN in U937 cells. In Caco-2, it increased NI and [Ca2+]i, but decreased NA, PMP, MMP and CN. Phospholipidosis was not observed in either cell line. pDMAEMA (10 mg/ml) did not induce any histological damage on rat colonic tissue and only mild damage to ileal tissue following exposure for 60 min. In conclusion, HCA reveals that pDMAEMA induces cytotoxicity in different ways on different cell types at different concentrations. HCA potential for high throughput toxicity screening in drug formulation programmes.
2010-08-01T00:00:00ZSafety and efficacy of sodium caprate in promoting oral drug absorption : from in vitro to the clinichttp://hdl.handle.net/10197/2908
Safety and efficacy of sodium caprate in promoting oral drug absorption : from in vitro to the clinic
Maher, Sam; Leonard, Thomas W; Jacobson, Jette; Brayden, David James
A major challenge in oral drug delivery is the development of novel dosage forms to promote absorption of poorly permeable Class III drugs across the intestinal epithelium.
To date, no absorption promoter has been approved in a formulation specifically designed for oral delivery of Class III molecules. Promoters that are designated safe for human consumption have been licensed for use in a recently approved buccal insulin spray delivery system and also for many years as part of an ampicillin rectal suppository. Unlike buccal and rectal delivery, oral formulations containing absorption promoters have the additional technical hurdle whereby the promoter and payload must be co-released in high concentrations at the small intestinal epithelium in order to generate significant but rapidly reversible increases in permeability. The most advanced promoter in the clinic is the medium chain fatty acid (MCFA), sodium caprate (C10) , a compound already approved as a direct food additive. We discuss how it has evolved to a matrix tablet format suitable for administration to humans under the headings of mechanism of action at the cellular and tissue level and in vitro and in vivo efficacy and safety studies. In specific clinical examples, we review how C10-based formulations are being tested for oral delivery of bisphosphonates using Gastro Intestinal Permeation Enhancement Technology, GIPET (Merrion Pharmaceuticals, Ireland) and in a related solid dose format for anti-sense oligonucleotides (ISIS Pharmaceuticals, USA).
2009-12-17T00:00:00ZRestoration of rat colonic epithelium after in situ intestinal instillation of the absorption promoter, sodium capratehttp://hdl.handle.net/10197/2761
Restoration of rat colonic epithelium after in situ intestinal instillation of the absorption promoter, sodium caprate
Wang, Xuexuan; Maher, Sam; Brayden, David James
Sodium caprate (C10) is an oral absorption promoter that is currently in clinical trials as a component of solid dosage forms for poorly permeable small molecules and peptides. Clinical data with zoledronic acid tablets suggest that significant delivery along with acceptable safety can be achieved from a once-a-week dosing regime. C10 has surfactant-like properties at the high doses used in vivo and therefore we examined its effects on rat intestinal epithelium following intestinal instillation. Results: Addition of 100 mM concentrations of C10 with the paracellular flux marker, fluorescein isothiocyanate-dextran 4 kDa, permitted a bioavailability of 33% to be achieved. When C10 was added 10, 30 and 60 min in advance of fluorescein isothiocyanate-dextran 4 kDa, enhancement still occurred, but was progressively reduced. Histology revealed that the permeability increase was likely related in part to superficial epithelial damage caused in the first few minutes of exposure, which was rapidly repaired within 30–60 min. Conclusions: Design of optimized dosage forms containing C10 should co-release the payload and promoter close to the epithelium in high concentrations. While C10 induces some epithelial damage, its remarkable capacity for epithelial repair may render this effect insignificant in vivo.
2010-07-01T00:00:00ZAntibacterial effects of poly(2-(dimethylamino ethyl)methacrylate) against selected Gram-positive and Gram-negative bacteriahttp://hdl.handle.net/10197/2755
Antibacterial effects of poly(2-(dimethylamino ethyl)methacrylate) against selected Gram-positive and Gram-negative bacteria
Rawlinson, Lee-Anne Betty; Ryan, Sinead M.; Mantovani, Giuseppe; Syrett, Jay A.; Haddleton, David M.; Brayden, David James
Antimicrobial coatings can reduce the occurrence of medical device-related bacterial infections. Poly(2-(dimethylamino ethyl)methacrylate)) (pDMAEMA) is one such polymer that is being researched in this regard. The aims of this study were to (1) elucidate pDMAEMA’s antimicrobial activity against a range of Gram-positive and Gram-negative bacteria and (2) to investigate its antimicrobial mode of action. The methods used include determination of minimum inhibitory concentration (MIC) values against various bacteria and the effect of pH and temperature on antimicrobial activity. The ability of pDMAEMA to permeabilise bacterial membranes was determined using the dyes 1-N-phenyl-naphthylamine (NPN) and Calcein-AM. Flow cytometry was used to investigate pDMAEMA’s capacity to be internalised by bacteria and to determine effects on bacterial cell cycling. pDMAEMA was bacteriostatic against Gram-negative bacteria with MIC values between 0.1–10 mg/ml. MIC values against Gram-positive bacteria were variable. pDMAEMA was active against Gram-positive bacteria around its pKa and at lower pH values, while it was active against Gram-negative bacteria around its pKa and at higher pH values. pDMAEMA inhibited bacterial growth by binding to the outside of the bacteria, permeabilising the outer membrane and disrupting the cytoplasmic membrane. By incorporating pDMAEMA with erythromycin, it was found that the efficacy of the latter was increased against Gram-negative bacteria. Together, the results illustrate that pDMAEMA acts in a similar fashion to other cationic biocides.
2010-02-08T00:00:00Z